JPH0742121Y2 - Device for optically detecting stratification of a sample in the rotor of a centrifuge - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention relates to centrifuges. In particular, the present invention relates to an optical scanning system for dynamically tracking the progress of stratification in a sample during the centrifugation step.

SUMMARY OF THE PRIOR ART It is known to bring a sample cell to the rotor of a centrifuge and observe said sample cell during centrifugation. Specifically, in the prior art, the light source is collimated. The collimated light passes through the upper and lower windows and falls downward through the sample cell. The collimated light passes exactly parallel to the plane of the sample stratification at the sample point. The imaging system refocuses the image of the sample on the plane of the scanning slit. The light passes through an interference filter as it travels to the scanning slit. The photoelectric detector in the immediate vicinity of the scanning slit is
Dynamically allow detection of bands while centrifugation is in progress.

It is known to collimate light parallel to only the radius of a rotating rotor. This can increase the light intensity in the sample, making the layers of the sample more visible.

Differences from the Prior Art There are important differences between the prior art and the matters disclosed herein. Specifically, in the prior art, collimation occurs in the radial direction of the sample. As will be described below, the use of a toroidally curved and scoring mirror with two distinct curvatures allows for the above-mentioned illumination under chromatically classified light of selected frequency and high intensity. The ability to inspect the sample is gained. In this case, the diffraction gradient of the sample does not significantly affect the image of the scanned sample.

Statement of Challenges Modern centrifuge technology is
It is necessary to detect the stratification of the sample. Unfortunately, such samples are almost totally non-conductive. Efficient use of radiation is needed.

Moreover, not only the need for monochromatic scanning,
There is a need to change the frequency of monochromatic scanning. That is, the wavelength of irradiation at which the sample is examined must be changed. All this must be done in the time range during which sample processing is occurring in the centrifuge.
This time range includes the rotor rotating at a speed of 100,000 rpm.

Conventional optical scanning techniques are not sufficiently optical sensitive or accurate to track a sample, are too complex, and produce good collimation sufficient to achieve good spatial scanning results. It does not, and will not work when a high diffraction gradient is present in the sample, and will not work until below 200 nm (nanometers).

SUMMARY OF THE INVENTION A centrifuge sample is optically scanned during centrifugation.
The sample placed in the centrifuge rotor having cells with windows, preferably top and bottom windows, comprises:
The stratification and the separated layers are spun until they occur in the sample. Such a layer is exactly perpendicular to the centrifuge radius at the sample point and parallel to the centrifuge axis of rotation. The light source is collimated in only one plane. This plane is the sample plane, including the axis of rotation of the centrifuge and passing through the sample point. Collimated light rays pass through and through the layers of the sample, exactly parallel to the axis of rotation of the centrifuge. The light is collimated and chromatically filtered by a toroidal mirror having two curvatures (hereinafter referred to as a toroidal mirror or mirror). The cylindrical shape of the mirror for one radius of curvature provides the collimation. The mirror rulings or rulings for other radii of curvature provide for color classification. The light is chromatically filtered and is not collimated into a plane perpendicular to the sample plane. A slit scanner having a slit orthogonal to the sample plane traverses the width of the sample below the cell. The transverse slit scanner accurately detects the exact position of the layer within the cell.

A preferred optical path is disclosed that includes a high intensity flash point source in the 20,000 watt range. The point light source has a diameter
It is thrown through an aperture of 1 mm or less.
This light will be incident on the toroidal concave diffraction grating lying above the sample points of the cell. The grid includes a cylindrical portion on the sample surface. The grating, which also acts as a mirror, provides collimation in the sample plane exactly orthogonal to the layers formed in the rotating sample.

The curvature of the cylindrical part is such that no collimation results when the grating is rotated about its axis of rotation. The mirror is scored as a diffraction grating along a line perpendicular to the sample plane. The sample is
Upon passing the sample point of the cell, it is flashed by the light. Light from the light source impinges on the mirror overlying the cell and is chromatically classified. The light passes through the cell in the band of about 5 nm and reaches the lower slit scan detector with the underlying photodetector. The slit scanning detector in combination with the photoelectric detector accurately determines the position of the layers formed in the cell during centrifugation.

The mirror can be tilted on an axis contained in a plane containing the axis of rotation and the radius at the sample point. The tilt axis of the mirror is preferably orthogonal to the rotation axis of the rotor. The mirrors are provided with unequal spacing so that a simple tilt can be used to obtain the desired color output without the need to adjust the focal length of the mirrors with respect to the sample to obtain chromatically classified light. To supply. The result is an optical sampling system. This sampling system can tolerate only four decades of light attenuation, allowing the kinetics of centrifugation to closely track in the separated bands that require high resolution.

Other objects and advantages It is an object of the invention to increase the irradiation intensity in the sample being processed by spinning in a centrifuge. According to this aspect, an apertured light source is flashed at the moment a sample is to be taken. The light from the light source is incident on the mirror just above the sample. The mirror includes a surface, which is preferably a cylinder, on a surface including the rotation axis of the centrifuge and the radius of the sample point. Light is reflected downwards from the mirror and collimated exactly parallel to the axis of rotation of the centrifuge at a sample plane containing the sample point and the axis of rotation of the rotor. A slit scanning detector arranged at right angles to the sample surface has an excursion that moves back and forth below the sample.
This detector accurately determines the stratified layer by the respective optical absorption when the stratified layer is dynamically formed in the centrifugation step.

The advantage of collimating the scanning light only in the sample plane including the rotation axis of the rotor and the sample point is:
This means that irradiation from the light source is used more effectively. For example, if the sample were illuminated by a standard collimated beam, it is possible to illuminate the sample with 10 times the illuminance that would be available. In this case, the normally collimated beam includes collimation across the sample plane.

Yet another advantage of the scanning system disclosed herein is that the narrow bands can be dynamically tracked while the very narrow bands are sorted from the sample. The sedimentation process of the bands (eg their sedimentation coefficient) can be actively followed.

Yet another advantage of the disclosed collimation is that collimation reduces the need for an imaging system to refocus the cell on the plane of the image detector. This allows operation at a resolution in the steep gradient of the diffraction of the sample.

Yet another object of the invention is to disclose an apparatus which allows the sample to be chromatically scanned at different wavelengths during the centrifugation step. According to this aspect of the invention,
The mirror is scored and curved. The mirrors are preferably provided with regular unequal spaced ruled lines. These ruled lines are provided to eliminate the need to change the distance of the mirror from the sample, as the sampling wavelengths are different.

An advantage of this aspect of the invention is that simply tilting the mirror on which the light source is incident changes the wavelength of light scanning the sample.

Yet another advantage of this lined mirror is that the mirror keeps the efficient use of light in the sample by avoiding collimation of the light in the plane of the layers formed in the sample. is there. Increased illumination is maintained in the sample.

Yet another object of the present invention is to disclose a folded optical path having a long light path from the light source to the sample and a short light path from the sample to the detector.

An advantage of this aspect of the invention is that the sample is inspected in large depth fields with almost perfectly parallel rays despite the angular arrangement of the grating. At the same time, the sample once tested will be in direct contact with the detector. Almost no degeneration of the optical image of the sample with respect to the slit detector occurs.

BRIEF DESCRIPTION OF THE DRAWINGS Other objects, features and advantages of the present invention will become more apparent after referring to the following detailed description and attached drawings. In the drawings, FIG. 1 is a side view of a centrifuge incorporating an optical scanning system of the present invention, FIG. 2 is a plan view of the centrifuge of FIG. 1, and FIG. 3 is a mirror in which color dispersion is ruled. Fig. 4 is a side view taken in the plane caused by Fig. 4, Fig. 4 is an optical schematic diagram showing the arrangement of mirrors for scanning the sample into exactly parallel light rays, and Fig. 5 is the folding used by the sample of Fig. 1. FIG. 6 is an optical schematic view of the path of the reflected light, showing the resolution provided by the optical system shown here, and FIG. 6 is a cutaway side view showing the mechanism for rotating the mirror.

With reference to Figures 1 and 2, a so-called "ultracentrifuge" is shown. Briefly, the electric motor M rotates the rotor R around the axis A. High speed is required. Rotating rotor R at 100,000 rpm is not uncommon.
Centrifugation is performed in vacuum to avoid windage, as is well known in the art.

The sample is contained in cell S ₁ . The cell includes an upper window 20 and a lower window 22 that allow light to pass parallel to the axis of rotation of the rotor R. An optical sample formed in accordance with the teachings of the present invention is
It is through each window that performs the method of inspecting the stratification of the sample.

In terms of light, the tube T has a light source L which is transmitted as a quadrangular pyramid so as to hit the mirror M. The light from the mirror M is reflected downward, passes through the folded tube T _1, and passes through the window 2 of the cell S ₁ of the rotor R.
Pass 0 and 22. After passing through the respective windows 20, 22, the light is incident on the detector D (Fig. 2). As will be described later, the detector D is a movable slit capable of having an excursion over the radial length of the sample.

The forces generated by a centrifuge typically range from 5,000 to 500,000 gravitational fields. It is an object of this invention to inspect the dynamically formed layers as the centrifugation action continues. Discontinuing the centrifugation action to observe the sample may correctly destroy the result that is being observed,
Will be understood. Specifically, the stratification that can occur in the created gravitational field is often lost by diffusion when the gravitational field is removed.

For the following applications, it is necessary to define a surface. Once defined, these planes make it possible to discuss the arrangement of optics in a straightforward way.

First, the sample is typically located at sample point P when the sample is optically read for stratification. The sample point P is located along a radius 26 from the centrifuge axis of rotation A.

The sample plane includes an axis of rotation A and a radius 26 extending from the line axis through the sample point. This sample surface is the surface where light collimation occurs. The same sample surface is that of FIG.

It is also necessary to describe the plane orthogonal to the sample plane. This surface is a dispersion surface along which chromatic dispersion of light occurs. No collimation of light occurs at the dispersion plane. This dispersion plane is the plane of the drawing in FIG.

After describing each of these aspects, the special optical functions of the present invention can be explained. This function firstly involves the collimation of light to make the desired inspection of the layer along the sample plane. Second, the color classification of light is discussed with respect to the dispersion plane. Finally, referring to FIG. 5, the resolution characteristic of the present invention will be described.

Referring to FIG. 3, the strobe light source L passes through an aperture 28, which preferably has a diameter of 1 mm or less. The light 40 from the light source is the cylindrical diaphragm 30,
It has passed through 32 and is incident on the mirror M.

With reference to FIG. 4, the reader will understand that the light source L is not shown. FIG. 4 shows, however, that the slit detector diverges downwards from the mirror M, through sample 50.
Light 40 is shown passing through 52 and reaching detector D. It will be appreciated that slit 52 scans the underside of sample 50. In such a scan it will discriminate the layer exactly parallel to the axis of rotation A and perpendicular to the sample plane of FIG.

The mirror M shown here is provided in a cylindrical shape with respect to the light source L in the sample plane of FIG. This cylindrical shape is chosen so that the rays 40 are precisely collimated in the plane of FIG. 4 along a path parallel to the axis of rotation of the rotor R. Thus, every classified layer of sediment, such as the sediment present in band B, has a collimated ray 40 exactly parallel to and passing through band B.

The mirror M is formed in a shape along one axis for generating collimated light rays. Along the other axis, the mirrors are provided with different curvatures and differently spaced ruled lines so that the tilt of the mirrors changes color and produces light.

Such ruled lines are known. See Puchard et al., U.S. Pat. No. 3,909,134, issued Sep. 30, 1975. Additional relevant prior art relating to the construction of such mirrors is US Pat. No. 3,930,728, issued to Puchard et al., Issued Jan. 6, 1976, Mar. 2, 1973.
Non-Puchard US Patent No. 3,721,487 dated 0
No. 3,942,048, issued March 2, 1976, to U.S. Pat. No. 3,942,048, and U.S. Pat. No. 3,628,849, issued December 21, 1971.

Rotation of the grating to change the wavelength occurs about 30 ° from a straight line perpendicular to the light source. The effective curvature for collimation does not change regardless of the grating angle.

The reader will understand that the illustrated optics are preferred. Alternative optics can be used that collimate the light only into the sample plane. For example, a combination of lens and mirror may be used.

It will be seen that the slit 52 traverses the detector D back and forth along the path indicated by the double-headed arrow 54. In such a traversing motion, detector D will discern the difference in light reception, as described in Cohen US Pat. No. 3,712,742 issued Jan. 23, 1973.

Some numerical examples are valid. Specifically,
Light source L is typically a strobe xenon source. At the moment of flashing, the light source contains an output in the range of 20,000 watts.

A large amount of light attenuation can be caused by an essentially non-conductive layer such as band B in sample 50 in cell S ₁ . A total of 17 decades (10 ¹⁷ ) of light attenuation can occur. Light attenuation in the sample is 3 decades (10 ³ )
Can be.

Returning to FIG. 3, it is also desirable to scan the sample 50 in cell S ₁ by the color-sorted bands. For example, it is desirable to scan proteins in the centrifuge that fall into the 200-800 nm range or higher, which range is in the ultraviolet and is the visible portion of the optical spectrum. Therefore, the mirror M has a curvature with unequal spacing ruled lines in the plane of FIG. As can be seen in FIG. 3, said ridges extend in and out of the plane of the figure. Align the mirror with the axis 60, as indicated by arrow 62.
By rotating around, the scan of sample 50 is 5 nm
Can be done in bands of width. The reader is advised that the scanning or pass width of the optical band is virtually determined by the fixed angle for the radiation of the mirror defined by the windows 20, 22, as seen in FIGS. 3 and 4. Will understand.

Serendipity will exist from such scans. Specifically, the bands of light that are chromatically classified are parallel to and within the plane of Band B, as shown in FIG. In such an alignment, the band B and the respective faces of the band are illuminated by light. The illuminating light is on the order of 10 times the available light collimated for both planes of FIGS. By limiting the collimation to the plane of FIG. 4 and allowing chromatographic classification across band B in the plane of FIG. 3, more than 10 times more light reaches band B of sample S. To do.

Referring to FIG. 5, an optical schematic of the system is shown. This schematic shows how the length of the folded light path shown in FIGS. 3 and 4 helps the band resolution of the sample.

It will be appreciated that the distance X ₁ represents the effective length of the optical path between the light source L and the sample S ₁ . This distance is equal to about 33 cm.

Similarly, the distance X ₂ is the distance between the slit 52 and the sample S ₁ . This distance is about 2 cm. It will be observed that the distance X ₁ of the folded light is much longer than the path X ₂ . Therefore, slit 52 will see band B in high resolution. here, Is.

It is emphasized that it is desirable to rotate the mirror M to obtain different color resolutions. The actual device for effecting this rotation is shown in FIG.

Referring to FIG. 6, there is shown the optics tube in the vicinity of the mirror shown schematically in FIGS. The mirror is the fourth
It is shown in the same plane as the schematic of the figure.

Specifically, the light passes above the tube T. An optical baffle causes the light source L (first
Decrease all parts of the beam from the figure). The light is reflected from the mirror M and passes down the tube T ₁ to sample the contents of the rotating cell.

The mirror M rotates about the pivot 102. The mirror is a gear 1
A gear train including 03, 104 provides such rotational movement. The gear train also includes reduction gears 105, 106 and an idler gear 107, the idler gear 107 ultimately being a rack.
Driven by 108. As will be readily appreciated, linear movement of the rack 108 toward and away from the mirror may result in finely adjusted and precise rotation of the mirror M. Since the mirror is always collimated, changes in the angle of the mirror only affect the color being sampled in the spinning cell.

The reader will appreciate that the ability to change colors quickly allows for rapid adjustment of color to sample the classified material within the cell. in this way,
During the passage of the photodetector over the radius of the individual cells, the color of the sampling light can be changed rapidly due to the optimal detection of the classified layers with varying optical density.

The alignment of the slit 52 and the detector D as shown in FIG. 4 has the additional advantage which is not obvious. Specifically, as the sample S ₁ increases in the sorted state, various components of the sample will collect in the separated layers. These ingredients include various salts.

Unfortunately, such heavy components, especially those classified as salts, have different index of refraction. It will be appreciated that this is the case, but in order for slit 52 to find these bands, an opening of some angular width must be provided.

Therefore, slit 52 has an effective width of approximately 0.1 mm and is spaced relative to the effective surface of detector D, resulting in a plus or minus 2 ° from the vertical view of the sample. Using this technique, the light refracted by the sample with different refraction indices can still be observed. And deviated light exceeding this angle does not hit the detector.

 ─────────────────────────────────────────────────── --Continued front page (56) References JP-A-57-61934 (JP, A) JP-A-54-43082 (JP, A) US Patent 3652860 (US, A) US Patent 3909134 (US, A) US Patent 3628849 (US, A)

Claims (5)

[Scope of utility model registration request] 1. A centrifuge rotor for rotation about an axis, the chamber having at least two windows for allowing light to pass through the sample parallel to the stratification of the sample in the rotor at an observation point. Optically stratify the sample in the rotor of the centrifuge while the rotor is rotated to move the sample past the sample points on a sample plane that includes a sample axis and a rotational axis of the rotor that defines the sample axis. For detecting the sample through the window while the sample is being rotated by the rotor of the centrifuge, a first radius of curvature and a second radius of curvature. A toroidal mirror having means for collimating the light source parallel to the axis of the rotor and only on the sample surface, wherein A single toroidal mirror with collimated light passing parallel to the stratification and scribed with respect to the second radius of curvature to give a color classification of different frequencies at different angles with respect to the toroidal mirror; Means for rotating the toroidal mirror about the first radius of curvature to select the frequency of light for inspecting the sample, and after passing through a cell in the window to determine the stratification of the sample Means for optically detecting the stratification of the sample in the rotor of the centrifuge, including means for detecting light. 2. The apparatus of claim 1 including means for moving the means for detecting to inspect the stratification. 3. The apparatus of claim 1, wherein the two windows in the rotor are a top window and a bottom window. 4. A combination of centrifuges for sorting said samples to form a layer from a sample, the rotor of the centrifuge being rotated about an axis of rotation, the light being parallel to said layers. A rotor having a first window and a second window for passage therethrough and defining a chamber holding the sample away from the axis of rotation of the rotor, and through the chamber for determining layers A glowing light source that passes through a sample plane that includes the rotation axis of the rotor and a sample point, and means that defines a path of light through the window of the rotor for detection of the layer. A detector for detecting light passing through the sample, whereby the layer is observed, wherein the means for defining the path of light is a single toroidal mirror having two radii of curvature, curvature A toroidal mirror having one of the diameters formed to limit collimated light only to the sample surface, and the other of the radii of curvature being scored to provide light of different frequencies at different angles from the toroidal mirror; While maintaining collimation of light in the sample,
Means for rotating the toroidal mirror with respect to the first radius of curvature for selecting a frequency of light for scanning the sample. 5. The centrifuge combination of claim 4, wherein the toroidal mirror is scored by diffraction scribing lines parallel to the sample surface.